SOD catalyzes dismutation of superoxide radical (hereinafter, referred to “O2−.”) into hydrogen peroxide and oxygen as per the following redox reaction:2O2−.+2H+=H2O2+O2 This reaction is the first enzymatic cellular defense against oxidative stress caused by O2−.. O2−. is generated by a number of metabolic perturbations and hence SOD has implications in all those reactions, wherein O2−. is produced in the amount leading to cellular injury. According to the U.S. Pat. Nos. 6,485,950 and 7,037,697, we have extracted an autoclavable copper/zinc superoxide dismutase (hereinafter referred as Cu/Zn SOD) from Potentilla atrosanguinea (hereinafter, referred to “Potentilla”) which shows activity at sub-zero temperatures. In yet another U.S. patent application Ser. No. 12/315,301, we have cloned Cu/Zn SOD gene from Potentilla in Escherichia coli (hereinafter, referred to “E. coli”). Analysis of Cu/Zn SOD gene sequences from various sources showed maximum variability at 3′ and 5′ regions with least variability in the middle part of the gene and that can be utilized to synthesize degenerate primers for amplification of Cu/Zn SOD genes. Degenerate primers have been designed from the selected conserved regions of the sequences and used for amplification of Cu/Zn SOD gene from diverse plant species.
Below is given a state of the art knowledge in relation to oligonucleotide primers for amplification of Cu/Zn SOD gene from various sources
Reference may be made to document (1) by Liu, J. J., Goh, C., Loh, C., Tay, E. B. H. and Pua, E. C. (Plant Physiol., 1998.116: 867) wherein cDNAs encoding Cu/Zn SOD were cloned from Brassica juncea, using degenerate oligonucleotide primers, synthesized from conserved domains of plant Cu/Zn-SOD homologs. However, these forward and reverse primers corresponded to the polypeptides of GC(M/I)STGPH (SEQ ID NO:45) and NAGGR(L/V)AC (SEQ ID NO:46), respectively. These primers were used for amplification of DNA from mustard cDNA library using PCR and an expected 300 bp DNA fragment was amplified.
Reference may be made to document (2) by Banks, G. K., Robinson, A. S., Kwiatowski, J., Ayala, F. J., Scott, M. J. and Kriticou, D. (Genetics, 1995. 140: 697-702) wherein four degenerate primers were used to amplify Cu/Zn SOD from Medfly (Ceratitis capitata), which were synthesized from three conserved regions of the known SOD enzyme. However, primer I, II, IIa and IIIb corresponded to the polypeptides of HGFHVH (SEQ ID NO:47), GPHFNP (SEQ ID NO:48), GCGVIG (SEQ ID NO:49), ACGVIG (SEQ ID NO:50) respectively. A 330 bp DNA fragment was amplified with primers I and Mb, while 260 bp fragment was obtained with primers II and IIIb.
Reference may be made to document (3) by Saavedra, N. Y. H., Egly, J. M. and Ochoa, J. L. (Yeast, 1998. 14: 573-581) wherein degenerate primers were used to amplify Cu/Zn SOD from Marine Yeast (Debaryomyces hansenii), synthesized from the Cu/Zn SOD protein sequence from yeast (Saccharomyces cerevisiae). N-terminal peptide sequences (VSGVVNFEQSSEEDPT (SEQ ID NO:51)) obtained from pure Debaryomyces hansenii Cu/Zn SOD protein showed 81.5% homology with the reported Saccharomyces cerevisiae Cu/Zn SOD sequence and hence the N- and C-terminal sequences of the Saccharomyces cerevisiae Cu/Zn SOD nucleotide sequence were used to design the degenerate primers (NT1: ATGAA(AG)GCIGTITG (SEQ ID NO:52) (TC)GTIATGACIGG (SEQ ID NO:53) and CT1: TC(AG)TC(TC)TC(AG)TT(TC)TC(AG)TG(GTA)AT(I)ACCAT) (SEQ ID NO:54). An amplified PCR product of 470 bp was obtained using degenerate primers NT1 and CT1.
Reference may be made to document (4) by Plantivaux, A., Furla, P., Zoccola, D., Garello, G., Forcioli, D., Richier, S., Merle, P. L., Tambutte, E., Tambutte, S. and Allemand, D. B (Free Radical Biology & Medicine, 2004. 37:1170-1181) wherein degenerate primers were used to clone two Cu/Zn SOD from sea anemone. Degenerate primers (CuF: GCNGGNCCNCAYTTYAAYCC (SEQ ID NO:55) and CuR: CCRCANGCNARNCKNGCNC (SEQ ID NO:56) CNCGRTTNCC (SEQ ID NO:57)) were designed from the highly conserved regions of the Cu/Zn SOD amino acid sequences from phylogenetically different organisms. These primers were used in RT-PCR and two fragments of approximately 250 bp were amplified.
The Drawbacks are:
                1. The oligonucleotide primers reported so far amplified very short region of the gene which do not cover the metal binding domain an essential requirement for the activity of Cu/Zn SOD.        2. The primers which amplify the gene including copper/zinc binding domain were not universal and hence could not be used to amplify the gene.        3. There is no report to show that partial genes cloned through degenerate primers will have SOD activity.        4. There is no report available about isolating the partial Cu/Zn SOD gene from plant species; Camellia sinensis, Caragana jubata, Arnebia euchroma, Rheum emodi, Picrorhiza kurrooa, Stevia rebaudiana, Curcuma aromatica, Eragrostis atrovirens, Echinocloa crussgalli, Eleucine indica, Cynodon dactylon, Pennisetum clandistinum, Toona sinensis and Lantana camara.         5. Apart from Potentilla atrosanguinea no other SOD has been reported which is autoclave stable        6. No Cu/Zn SOD enzyme except from Potentilla atrosanguinea, has been reported to function at sub-zero temperature.        7. There is no Cu/Zn SOD gene that is isolated from Caragana jubata and Curcuma aromatica, and made to express in E. coli.         